Aberrant gene transcription is a key hallmark of cancer, and the notion of “transcriptional addiction” has been posited in MYC-driven malignancies and MLL-rearranged acute myeloid leukemia (AML). This transcriptional vulnerability may be targeted therapeutically with inhibitors of transcription regulators, including Cyclin-dependent kinases (CDK) 7, 9, 11, 12 and 13.
Using THZ531, a covalent inhibitor targeting CDK12 and 13, we investigated the molecular, biological and therapeutic consequences of inhibiting CDK12/13 in MLL-rearranged AML. THZ531 potently induced apoptosis and downregulated RNA polymerase II (Pol II) C-terminal domain (CTD) Ser2 phosphorylation. To dissect the individual functions of CDK12 and 13, we utilized CRISPR-mediated homology-directed repair to create AML cells containing ATP analog-sensitive CDK12, CDK13 and CDK12/13 alleles. Using this system, functional redundancy between CDK12 and 13 was apparent for the maintenance of cell survival and Pol II phosphorylation. Apoptosis and decreased Ser2 phosphorylation were only observed following combined inhibition of both kinases, phenocopying the effect of THZ531.
To identify the molecular responses to CDK12/13 inhibition, extensive next-generation sequencing based techniques revealed that CDK12/13 inhibition globally perturbs the transcriptional landscape in a manner that is distinct from inhibition of other transcriptional CDKs. Importantly, CDK12/13 inhibition resulted in a marked increase in alternative last-exon events, a genetic event that has been linked to a decreased rate of Pol II-driven transcriptional elongation. ChIP-Seq analysis for total Pol II and pS2-CTD revealed that CDK12/13 inhibition resulted in blocking the processivity of Pol II along the gene body, concurrent with loss of gene-body Pol II Ser2 phosphorylation. Furthermore, PRO-Seq analysis demonstrated that the velocity of Pol II elongation was greatly reduced upon CDK12/13 inhibition.
Our study has identified the specific roles of CDKs 12 and 13 in regulating transcription elongation rates and the inclusion of alternative last exons and highlights a potential therapeutic pathway to target transcription-addicted malignancies.